The separation and selection of particles, in particular living cells such as spermatozoa, is the subject matter of many methods. In particular, the qualitative and quantitative, but also cost-effective separation and selection of spermatozoa, the so-called “sexing”, plays an important role here. In this case, this relates to the sex-specific sorting of spermatozoa, which is assigned great economic significance in the field of livestock.
In the field of livestock breeding and livestock reproduction, in particular of cattle, but also of pigs, sheep, poultry, or horses, the prior establishment of the sex of the offspring is advantageous. Flow cytometers, as are commercially available, are conventionally used for the sex-specific sorting of sperm. To differentiate the X and Y chromosomal sperm, differences in the DNA content of the two types of sperm in comparison to one another are utilized, which can be determined by special detection capabilities of flow cytometers. On the basis of the known differences in the DNA content between sperm which contain the larger X-chromosome and those which contain the smaller Y-chromosome, for example, in the case of cattle by approximately 4%, it is possible to utilize this difference in the DNA content to determine the sex of the sperm, on the one hand, and to separate and select these sperm, on the other hand.
Fundamentally, the particles to be detected and selected are isolated in a fluid stream in flow cytometry, to then be selected accordingly on the basis of a determined signal. For this purpose, this is conventionally the signal of a DNA-specific fluorescent pigment, which is detected with the aid of excitation via a laser beam. Further desired particle properties, such as size, etc., can be determined with the aid of the detectors. This is conventionally performed via the determination of the scattered light of the incident laser light. With the aid of the detectors, corresponding measuring signals are generated, which permit a statement about the DNA content of the particles and also about the size and other properties of the particles. Separation and selection of the particles, in particular the sperm, is then carried out via a sorting device. A corresponding separation and selection is conventionally performed via a deflection in an electrical field of the droplets produced from a fluid stream. For this purpose, the fluid stream and therefore also the droplets produced therefrom are electrically charged to then be electrostatically deflected and selected accordingly during the passage through an electrical field.
In the case of sperm, the selection is performed based on the DNA content. The determination of the DNA content is also strongly dependent on the morphology of the cells, however, so that the orientation of the sperm can have an influence on the measured value of the DNA content.
Accordingly, it is necessary to provide those methods which permit an orientation of the cells, in order to improve the determination of the DNA content.
Improved nozzles for flow cytometers are described in WO 01/40765. Damage to the cell is to be prevented by the nozzle geometry described therein.
WO 2005/075629 describes a device for measuring oriented aspheric cells. For this purpose, a determination of the orientation of the sperm is carried out via a second light source.
Furthermore, nozzles or functional elements such as the injector for the core jet for producing a fluid stream are described, which do not have a round formation, but rather an ellipsoidal formation at the outlet.
To obtain the highest possible yield during the selection, the sperm are aligned such that the flat side of the sperm head is aligned perpendicular to the radiation to be detected. For this purpose, a specially designed nozzle geometry is used. This nozzle geometry permits the core jet containing the sperm to be focused hydrodynamically and simultaneously shaped elliptically by an externally enclosing, liquid envelope stream. The shaping is based on the principle that by way of the elliptical formation of the nozzle inner wall, which tapers toward the outlet, and the asymmetrical radial velocity gradients of the envelope stream, the core jet is also elliptically shaped and focused. In principle, the ellipsoidal shape of the core jet cross-section is thus applied pivoted by 90° in relation to the ellipsoidal shape of the nozzle wall. This results in a low sorting rate.
The object of the invention is to provide novel devices having an internal and an external functional element for producing a fluid stream, in particular nozzles for flow cytometers, which permit a significantly increased sorting rate of particles, in particular living cells, such as spermatozoa.